Mass spec and reproducible experiments

Liquid chromatography-mass spectrometry (LC/MS) is the workhorse of today's protein analysis and just as the science seems to leap forward every other day, so does the technology.

In November last year, Agilent Technologies launched its new quadrupole time-of-flight (Q-TOF) LC/MS system, which it says offers greatly improved mass accuracy, sensitivity and speed for a range of complex experiments, including proteomics and metabolomics.

Very kindly, Agilent has donated one of its new machines to the laboratory of Dr Bruno Domon at the Institute of Molecular Systems Biology at the Swiss Federal Institute of Technology (ETH) in Zurich. Here, Domon and his colleagues are putting the new Q-TOF through its paces, doing intense testing of the machine along with Agilent's HPLC chip systems.

Domon, who works with Professor Ruedi Aebersold, one of the best known scientists in the field of proteomics, was in Australia in February for the Lorne Protein Structure and Function conference and held a workshop on one of the biggest challenges in proteomics today - how to run reproducible experiments in order to identify biomarker candidates, in particular glycan biomarkers.

"The main idea of the technology we are working with is the ability to move away from the standard shotgun approach to proteomics, where you hope to get as many peptides and proteins as possible," Domon says.

"There is a big limitation to that approach. I use the metaphor of an iceberg - with the shotgun approach, you only see the tip, not what is under the water. The ultimate goal is to get more sensitivity in a more targeted way."

Last year, Domon, Aebersold and their colleague Paola Picotti published a paper in Molecular & Cellular Proteomics on the implications of the proteolytic background for shotgun proteomics. They found that the number of peptides observed from a single protein is at least one order of magnitude greater than previously assumed.

"This unexpected complexity of proteomic samples implies substantial technical challenges, explains some perplexing results in the proteomic literature and prompts the need for developing alternative experimental strategies for the rapid and comprehensive analysis of proteomes," the researchers write.

What Domon and his team are trying to do now is to approach the LC/MS not just as a mass spec but as a tool to run protein samples in a reproducible way. They are testing Agilent's Q-TOF - and other companies' technology - to develop a new workflow strategy for proteomics researchers that will ensure greater accuracy in experiments.

Agilent will have one of its new Q-TOF mass specs on show at the AOHUPO/PRICPS conference in Cairns this week, and is also sponsoring another international proteomics expert to attend, Dr Christopher Gerner from the Institute of Cancer Research at the Medical University of Vienna. He will talk about establishing a database of another of the 'omes' - the secretome, the full complement of proteins that are secreted from the cell.